A key event in the regulation of eukaryotic gene expression is the post- translational modification of nucleosomal histones, which converts regions of chromosomes into transcriptionally active or inactive chromatin. The most common post-translational modifications of histones is the acetylation of epsilon-amino groups on conserved lysine residues in the histones' amino-terminal tail domains. Hyperacetylation of histones generally correlates with transcriptionally active chromatin, perhaps by increasing the accessibility of transcription factors to nucleosomal DNA, while hypoacetylation of histones correlates with transcriptional silencing. Although significant advances have been made toward understanding histone acetylation, much less is known concerning histone deacetylation. For example, during the last few years, several genes that encode histone acetyltransferase enzymes have been identified and extensively characterized. In contrast, the few mammalian proteins that have been found to possess histone deacetylase activities have not yet been characterized at all. In addition, while much effort has been devoted to understanding mechanisms that mediate histone acetylation, there are gaps in our knowledge concerning mechanisms that govern histone deacetylation. Three mammalian histone deacetylases (HDAC1, HDAC2, and HDAC3) with homology to the yeast protein RPD3 have recently been cloned. In this proposal, the overall hypothesis is that histone deacetylases play a pivotal role in the regulation of gene expression. The goal of this project is to understand the underlying mechanisms of histone deacetylation, with particular emphasis on a detailed dissection of the mammalian RPD3-related HDACs. These studies will ultimately provide important insights critical for a thorough understanding of the intricate mechanisms operation to orchestrate gene expression in mammalian cells.